The present invention is directed to a multicapillary fluorescent detection system, and more specifically, to a multicapillary multilaser detection system.
Capillary electrophoresis (“CE”) has found widespread application in analytical and biomedical research and has been employed for the rapid separation and analysis of charged species including synthetic polynucleotides, DNA sequencing fragments, DNA restriction fragments, amino acids, optical isomers of dansyl amino acids, and the separation of proteins, viruses and bacteria. Micellar electrokinetic capillary chromatography, isoelectric focusing, and on-column derivatization can all be performed on CE columns.
The advantages of CE arise from the use of a small inside diameter (20-200 μm) capillary. Very high electric fields can be applied along small diameter fused-silica capillaries. Since the electrophoretic velocity of the charged species is proportional to the applied field, CE can achieve rapid, high-resolution separation. Considerable heat is generated by Joule heating. However, the large surface-to-volume ratio of the capillary channel and the use of thin capillary walls (50-150 μm), allows rapid heat dissipation when used in connection with cooling systems.
Automated DNA sequencing has gained widespread attention in recent years. Current methods for sequencing strands of DNA typically apply Sanger-Coulson type chemistries and electrophoretic methods to separate the DNA fragments generated during the sequencing reaction. Because capillary electrophoresis and particularly CE combined with laser induced fluorescence (CE-LIF) detection offers rapid charged species analyte separations and high detection sensitivity, it is particularly attractive as a separation technique in DNA sequencing applications. In order to take advantage of laser induced fluorescence some current DNA sequencing reactions involve fluorescently labeling DNA fragments and then separating and detecting the sequencing reaction products using CE-LIF techniques.
Even though CE separations are rapid, the throughput associated with CE based DNA sequencing is generally less than that of conventional slab gels when only one capillary forms the separation system. In order to overcome this limitation it has been suggested that multiple capillaries be used in parallel to achieve the desired throughput. Of course, the increased throughput of a multiple capillary CE system becomes a costly and cumbersome system when used in combination with a multiplicity of discrete source and detector elements. Moreover, the discrete source/detector element approach also becomes much more complicated when the requirement for multiple wavelength monitoring is added.
A multiple capillary CE-LIF system which utilizes a confocal fluorescence scanner is described in U.S. Pat. Nos. 5,091,652 and 5,274,240. These scanners rely on moving continuously each capillary in an array of capillaries across the light path of a laser. Alternatively, it has been suggested that the whole optical head be moved across the array of capillaries in a “sweep” scan (HPCE Meeting, San Diego, 1994). Both of these approaches require the movement of relatively heavy system components as one capillary is moved from the light source and the next capillary is moved into the light source. Necessarily, a large amount of time is consumed in moving the system components. It is likely that valuable separation information may be missed as a result of the lag time inherent in these systems. Moreover, the detection sensitivity attributed to fluorescence systems are somewhat compromised since the light source does not reside on an optimal part of the sample volume contained in each capillary, but is continuously scanned across the capillary.
Furthermore, since relatively heavy components are being moved in the prior art multicapillary detection systems, it is likely the momentum of the moving machinery will result in a gradual misalignment of the capillaries with respect to the light source or with the detector. Like the time delay problem, misalignments may lead to the loss of information and/or decreased sensitivity and increased detection limits. Also the motors and mechanisms required to move the capillaries necessarily result in additional cost associated with producing the scanner.
A multicapillary CE-LIF system utilizing a scanning mirror and parabolic reflector is described in U.S. Pat. No. 5,675,155, the entire contents of which are hereby incorporated herein by reference. This system relied on a spinning filter wheel in combination with a single detector for detection of multiple dyes. However, this system allows only a single dye to be detected at any time, and because of the need to switch filters only allows for a limited signal integration time. Additionally, because the filter elements are placed within the filter wheel, and the rotational velocity cannot be altered during the course of a run, the relative integration time for detection of the fluorophores is fixed for a given filter wheel. Changing filter wheels is an expensive and complex task.
Accordingly, it is desirable to provide an economical, flexible, highly sensitive, stable and rugged detection system for use in connection with high throughput separation systems. It is further desirable to provide an automated detection system for use in connection with multi capillary CE-LIF systems. Such a system should have the capability of providing multiple excitation wavelengths and detecting multiple emission wavelengths.
There is therefore a need for a detection apparatus that solves the shortcomings of the prior art.